1 ethyl 3 dimethylaminopropyl carbodiimide edc

Cyanogen bromide activated Sepharose 4B (CNBr) and butyl Sepharose CL-8B were purchased from General Electric (Upsala, Sweden). Agarose 10BCL (50–150 μm) was purchased from Agarose Bead Technologies (Madrid, Spain). Fully activated glyoxyl–agarose 10BCL (150 μmol aldehyde groups/g) was prepared as previously described [30 (link)]. Thermomyces lanuginosus lipase (TLL), Candida antarctica lipase sp. 99–125 (CAL), 1,2-ethylenediamine (EDA), Ethanolamine hydrochloride, sodium metaperyodate, 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), Triton X-100, dithiotreitol (DTT), anthranilic acid (AA), methyl anthranilate (MA), aniline (AN), ethanol, p-nitrophenyl butyrate (p-NPB), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) and salts for buffering solutions were purchased from Sigma Chem. Co. (St. Louis, MO, USA). The sardine oil was a gift from BTSA, Biotecnologías Aplicadas, S.L. (Madrid, Spain); the Novozyme^® 435 was a gift from Novozymes (Bagsværd, Denmark). Geobacillus thermocatenulathus lipase 2 (BTL2) expressed in E. coli was produced, purified, and aminated in solid phase as previously described [22 (link)]. Other reagents and solvents were of analytical or HPLC grade. Novozyme^® 435 and Lewatit^® VP OC 1600 were kindly donated by Novozymes A/S and Lanxess^®, respectively.

Sodium alginate (M_w = 200,000–300,000; FMC Biopolymer, Philadelphia, PA) was dissolved in a 2-(N-morpholino)ethanesulfonic acid (MES) buffer at room temperature (pH = 6.5, 0.3 M NaCl). A peptide with the (glycine)₄-arginine-glycine-aspartic acid-alanine-(serine)₂-lysine (G4RGDASSK) sequence (Anygen, Seoul, Republic of Korea) was added to the alginate solution in the presence of N-hydroxysulfosuccinimide (sulfo-NHS; Pierce, Rockford, IL) and 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC, Sigma-Aldrich, St. Louis, MO). The peptide-modified alginate was purified by extensive dialysis with distilled water for 5 days (M_w cut-off = 3,500) and activated charcoal treatment and then sterilized with a 0.22 μm filter. The degree of substitution (DS) of the peptide was determined with the number of peptides per 100 uronic acid residues in the alginate chain. In this study, the DS was 0.15 [7 (link)].

rAlb was kindly provided by Novozymes Biopharma (Frederiksberg, Denmark). Glutaraldehyde (GA) and 1-Ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), obtained from Sigma-Aldrich (St. Louis, Missouri, USA), were used as crosslinkers (CL). Phosphate buffered saline (PBS) tablets without calcium and magnesium were obtained from MP Biomedicals Inc. (Santa Ana, CA, USA).